High-capacity nickel-IMAC resin for His-tagged fusion protein purification.
Thermo Scientific HisPur Ni-NTA Resin is a high-capacity, high-performance nickel-IMAC resin for routine affinity purification of His-tagged fusion proteins.
The specially prepared support consists of beaded agarose derivatized with the nitrilotriacetic acid (NTA) chelation moiety and loaded with divalent nickel ions (Ni2+). The immobilized metal affinity chromatography (IMAC) resin provides exceptional binding capacity and performance for recombinant His-tagged protein purification.
HisPur Ni-NTA Resin is available in several package sizes of resin slurry, three sizes of centrifuge-ready columns, complete purification kits, two sizes of FPLC-ready chromatography cartridges, and 96-well filter plates for high through-put needs. With these options, high-yield His-tag protein purification is possible at nearly any scale.
High capacity – bind up to 60mg of 6xHis-tagged protein per milliliter of resin
Versatile – purify proteins using native or denaturing conditions
Compatible – use with Thermo Scientific Cell Lysis Reagents and a variety of buffer additives
Flexible – available in multiple formats including bulk resin, spin columns, chromatography cartridges and 96-well filter plates
Cost-effective – reuse the same batch of resin at least five times
Easy to use – pre-formulated buffers available for kit formats
The expression and purification of recombinant proteins is central to protein regulation, structure and function studies. The majority of recombinant proteins are expressed as fusions with short affinity tags, the most popular being the polyhistidine (6xHis) tag. The method used to purify recombinant His-tagged proteins is immobilized metal affinity chromatography (IMAC), consisting of chelating resins charged with either nickel or cobalt ions that coordinate with the histidine side chains.
HisPur Ni-NTA Resin effectively purifies high levels of overexpressed His-tagged fusion proteins from bacterial lysates, such as those that are attained with Thermo Scientific B-PER Bacterial Protein Extraction Reagents. The resin performs well in batch-binding and spin-column procedures at a variety of scales. Performance equals or exceeds popular Ni-NTA resins from other suppliers.
Thermo Scientific HisPur Ni-NTA resin is effective for purifying His-tagged proteins in batch-bind and spin-column formats with comparable results to another supplier's resin.Left panel: Bacterial lysate (10mg total protein) containing over-expressed 6xHis-annexin V was applied to 0.2mL of HisPur Ni-NTA Resin and purified by the batch-bind method. Center panel: Bacterial lysate (1.7mg total protein) containing over-expressed 6xHis-annexin V was applied to a HisPur Ni-NTA Spin Column (0.2mL) and purified by the spin method. Right panel: The same amount of lysate (1.7mg) was applied to Supplier Q's spin column and purified per the manufacturer's instructions. Gels lanes were normalized to equivalent volume. M = molecular-weight marker, L = lysate load, FT = flow-through and E = elution.
Thermo Scientific HisPur Ni-NTA resin performs as well or better than other suppliers' nickel resins. Bacterial lysate (12mg total protein) containing over-expressed 6xHis-green fluorescent protein (GFP) was applied to HisPur Ni-NTA Resin (0.2mL) and purified by the batch-bind method. The same amount of total protein was applied to Supplier Q, Supplier C, and Ni-IDA resins per the manufacturers' instructions. Gel lanes were normalized to equivalent volume. M = molecular-weight marker and L = lysate load.
HisPur Ni-NTA Resin is a high-quality, stable and resilient affinity support. Tests confirm that no decrease in performance occurs after at least five repeated uses. These data indicated that the resin is highly resistant to structural degradation or nickel ion leaching during normal use.
Thermo Scientific HisPur Ni-NTA Resin can be used at least five times without losing performance. Bacterial lysate (20mg total protein) containing over-expressed 6xHis-Protein L was applied to HisPur Ni-NTA Resin (0.2mL) and purified by the batch-bind method. Before each reuse, the resin was washed with 20mM MES buffer, 0.1M NaCl; pH 5 (1mL) and followed by a water wash (1mL). Gels lanes were normalized to equivalent volume. M = MW marker, L = lysate load, FT = flow-through and E = elution.
HisPur Ni-NTA Resin and HisPur Cobalt Resin are alternative forms of IMAC, the former using nickel and the latter using cobalt as the chelated metal ion responsible for His-tag binding. Ni-NTA resins have been the most common IMAC resin choice for 6xHis-tag protein purifications because of the four metal-binding sites on the chelate, which enables high-protein binding and low-metal ion leaching. Cobalt binds less strongly and therefore more discriminantly, enabling greater purity but usually less yield in His-tagged protein purification.
HisPur Ni-NTA and HisPur Cobalt Resins maximize yield and purity, respectively.Gel panels: Bacterial lysate containing over-expressed 6xHis-AIF2 (6mg total protein) or 6xHis-GFP (4mg total protein) was applied to HisPur Ni-NTA Resin (0.2mL) and purified by the batch-bind method. The same amount of total protein was applied to Ni-IDA and HisPur Cobalt Resins and purified as described in the instructions. Gels lanes were normalized to equivalent volume. M = molecular-weight marker, L = lysate load and FT = flow-through.
Table. Elution fractions were analyzed for protein content using the Coomassie Plus (Bradford) Protein Assay Kit (Part No. 23236). Purity was determined by analyzing the gel with densitometry software.